Dynamic localization and clustering of dendritic Kv2.1 voltage-dependent potassium channels in developing hippocampal neurons
Section snippets
Materials
Sprague–Dawley timed pregnant rats were purchased from Taconic Farms (Germantown, NY, USA). Horse serum was obtained from JRH Biosciences (Lenexa, KS, USA). Alexa 488 and Alexa 594 conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies were purchased from Molecular Probes (Eugene, OR, USA). Fluorescein 5-isothiocynate and Texas Red conjugated goat anti-mouse IgG1 and IgG2a secondary antibodies were purchased from Southern Biotechnology Associates (Atlanta, GA, USA).
Primary hippocampal neuronal cell cultures
Kv2.1 clustering differs between pyramidal neurons and interneurons
We showed previously that cultured hippocampal neurons express Kv2.1 potassium channels (Maletic-Savatic et al., 1995, Murakoshi and Trimmer, 1999). We double-stained embryonic hippocampal neurons after 14 days in vitro (14 DIV) culture with antibodies specific for Kv2.1 and for the neuronal marker microtubule-associated protein 2 (MAP2). This revealed that all of the neurons in the cultures exhibited robust Kv2.1 staining that was restricted to the soma and proximal dendrites. However, the
Discussion
Analyses of the expression and localization of both endogenous Kv2.1 and recombinant GFP–Kv2.1 in hippocampal neurons developing in culture revealed a stepwise progression of Kv2.1 localization. Kv2.1 expression is first detected in the ER, which is typical for the original biosynthetic pool of any plasma membrane protein. The ER is the site of subunit assembly, where for Kv channels four α subunits assemble into a channel protein complex (Papazian, 1999). Developmentally, staining for Kv2.1 in
Acknowledgements
The authors thank Joan Speh and Dr. Gail Mandel for use of the confocal microscope, and Drs. Matthew N. Rasband and Kenneth J. Rhodes for critically reviewing this manuscript. Supported by NIH Grants NS34375 and NS42225.
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